Pharmaceutical anti-infective composition for inhalation

ABSTRACT

An anti-infective composition for inhalation, containing, at least an effective amount of an antimicrobial aminoglycoside compound or a salt thereof; and an effective amount of a biofilm modifier which is a macrolide compound or salt thereof.

FIELD OF THE INVENTION

The invention relates to pharmaceutical compositions for the treatmentof lung infections caused by biofilm producing bacterial, fungal orviral pathogens.

The invention discloses inhaled combinations of at least one antibioticagent which is an aminoglycoside or a salt thereof and at least onebiofilm modifyer agent which is a macrolide or a salt thereof, for thetreatment of recurrent lung infections associated with a biofilm.

The compositions of the present invention are meant to be administeredlocally in the lungs of patients, said lung administration beingperformed using a dry powder inhaler system or a nebulizer.

The weight ratio of each of said antibiotic and said biofilm modifyermay be superior or equal to 10% of said dry powder inhaler. The weightratio between the aminoglycoside and the macrolide in the compositionsof the invention can be comprised between 0.2 and 5, and the totalamount of both active ingredients per pharmaceutical composition can becomprised between 1 and 50 mg for a total weight of dry powder percomposition being comprised between 1 and 100 mg.

BACKGROUND OF THE INVENTION

Antibiotic resistance and persistent infections refractory to per os orinjected treatments are a major problem in bacteriologicaltransmissions, resistance to eradication and ultimately pathogenesis.While the consequences of bacterial resistance and bacterialrecalcitrance are the same, there are two different mechanisms thatexplain the two processes.

-   -   Antibiotic/Antimicrobial Resistance. In the case of antibiotic        or antimicrobial resistance, biofilms provide the unique        opportunity for bacterial to reside in close proximity with one        another for long periods of time. This prolonged juxtaposition        of bacterial allows gene transfer between and among bacteria,        allowing the genes of resistance to be transferred to same or        different strains of bacteria to neighboring cells that are not        resistant. Consequently, a virulent cell can transfer its        virulence genes to a non-virulent cell, making it resistant to        antibiotics.    -   Antibiotic/Antimicrobial Recalcitrance. In the case of        antibiotic or antimicrobial recalcitrance, there are two        possible explanations, both of which involve the biofilm and        both of which may be operative simultaneously. While gene        transfer may occur, it is not a factor in recalcitrance.

Biofilms are matrix-enclosed accumulations of microorganisms such asbacteria (with their associated bacteriophages), fungi, protozoa andviruses that may be associated with these elements. While biofilms arerarely composed of a single cell type, there are common circumstanceswhere a particular cellular type predominates.

Biofilms are the most important primitive structure in nature. In amedical sense, biofilms are important because the majority of infectionsthat occur in animals are biofilm-based. Infections from planktonicbacteria, for example, are only a minor cause of infectious disease.

In summary, the biofilm formation consists of planktonic cells adsorbingonto a surface, experience phenotypic transformations and form colonies.Once the colonizing cells become established, they secreteexopolysaccharides that serve as the backbone for the growing biofilm.While the core or backbone of the biofilm is derived from the cellsthemselves, other components e.g., lipids, proteins etc, over time,become part of the biofilm. Thus a biofilm is heterogeneous in its totalcomposition, homogenous with respect to its backbone and heterogeneouswith respect to its depth, creating diffusion gradients for materialsand molecules that attempt to penetrate the biofilm structure.

The first of the explanatory mechanisms of resistance offered by biofilmis simply a physical phenomenon: the biofilm structures present abarrier to the penetration of antibiotics and antimicrobial agents and aprotective shroud to physical agents such as ultraviolet radiation.

Another biofilm resistance mechanism is based on biochemical ormetabolic principles. Just as the deep-seated bacterial are protectedfrom chemical and physical agents by the “barrier” effect of thebiofilm, the biofilm also acts as a barrier to nutrients that arenecessary for normal metabolic activity. Further, the nutrient-limitedbacteria are in a reduced state of metabolic activity, which make themless susceptible to chemical and physical agents because the maximaleffects of these killing agents are achieved only when the bacteria arein a metabolically active state. In addition, biofilms are linked toother virulence factors of pathogens (like efflux pumps or alginatesecretion).

In particular, biofilms constitute a growing problem for the treatmentof respiratory diseases associated with infection like cystic fibrosis,diffuse panbronchiolitis, exacerbation of chronic obstructive pulmonarydiseases, pneumonia, etc. . . . .

The treatments of those diseases with antibiotics become consequentlymore and more difficult due to the resistance offered by said biofilm.

Biofilms are associated with various bacteria, fungi and viruses amongwhich Pseudomonas aeruginosa and Staphylococcus aureus cause the mostdramatical consequences in the above cited respiratory diseases.

Whichever the mechanistic explanations for either resistance orrecalcitrance, the removal or disruption of the biofilm is a mandatoryrequirement for the successful treatment of the infection.

Aminoglycoside antibiotics are very active antimicrobial agents buttheir use has been limited because of their high frequence of seriousand irreversible adverse events associated with their use. The mostcommon and important toxicity are nephrotoxicity and ototoxicity.Aminoglycosides are usually administered by intra-venous injectionbecause they are very poorly absorbed by the oral route. Nevertheless, anebulized formulation of Tobramycin (TOBI®) to treat lung infections dueto P. aeruginosa in Cystic Fibrosis patients is available.

TOBI® presents the advantage to allow to treat lung infections locallywith a lower systemic exposure than the intravenous formulation and isthus responsible of less side-effects. However, due to low respiratoryfraction compositions administered through nebulisation (5 to 8% of thenominal dose), the nominal dose to administer (300 mg of Tobramycinb.i.d.) is still too high and can be responsible of a significantfrequency and/or severity of side-effects.

Although, aminoglycoside antibiotics are very effective against severalplanktonic bacteria, they are much less effective if not ineffectiveagainst the same bacterial which have formed a biofilm. This phenomenonof resistance of biofilms is also true for other antibiotics andrepresents a major public health concern. In some specific lung diseaseslike cystic fibrosis and diffuse panbronchiolitis, it is of majorimportance to dispose of compositions which are able to destroy,disorganize, inhibit the biofilm and/or able to prevent its formation.

Oral dosage formulations of macrolides antibiotics are widely used totreat, among others, respiratory infections like acute exacerbations ofchronic bronchitis, sinusitis, rhinopharyngitis, . . . .

While macrolides antibiotics are mostly available as oral dosage formscontaining several hundreds of milligrams of the antibiotic, no inhaledform has been available up to now, because amounts of hundreds of mg areimpossible to administer ambulatorily by inhalation through systems likedry powder inhalers or metered dose inhalers.

In summary, oral and intravenous antibiotic compositions used to treatbacterial infections are efficient not and safe against bacterialbiofilms responsible for lung infections or surinfections.

Consequently, there is an urgent need for efficient and safe antibioticcompositions, administered directly in the lung and able to treat lunginfections due to biofilms. The present invention discloses a dry powdercomposition for inhalation allowing to obtain a) a high pulmonary amountof aminoglycoside antibiotic and b) high dose of a biofilm modifyerwhich is selected from the group of macrolides and derivatives, which isefficacious against said biofilm when administered directly into thelungs.

STATE OF THE ART

Several inventors have already described attempts to act on bacterialbiofilms.

JP 726 7868 relates to a biofilm-removing agent containing a macrolideantibiotic at a low concentration, capable of removing biofilm formed byperiodontal pathogens, making a drug effectively penetrate to and act onan affected part and also suppressing the formation of the biofilm anduseful for self care. This is a removing agent of biofilm periodontalpathogens containing a macrolide antibiotic having a 14-membered ring,preferably belonging to an erythromycin, a clarithromycin, atriacetyloleandomycin or a roxithromycin. For administration, theantibiotic may be contained in e.g. a slow-releasing ointment or film orin a solvent such as a mouth-washing preparation. The dose of themacrolide antibiotic is preferably 0.5-10 mg/day, especially preferably1-5 mg/day.

WO 200 602 9893 provides the use of a compound selected from the groupcomprising an anthraquinone and a naphtoquinone, stereoisomeric forms,racemic mixtures, metabolites, esters or salts thereof, or mixturesthereof, and/or of at least one plant extract or active fraction thereofcomprising said compound for preventing and/or inhibiting biofilmformation. The present invention further relates to compositions forpreventing and/or inhibiting the formation of a biofilm, oral healthproducts and a method for preventing and/or inhibiting biofilmformation.

US 2005/0049181 A1 describes a synergistic antimicrobial composition forinhibiting biofilm formation includes an iron-sequestering glycoprotein,a cationic polypeptide and a chelating agent, or an iron-sequesteringglycoprotein and a chelating agent, or an iron-sequestering glycoproteinand a cationic polypeptide. Additionally, surfactants and quaternaryammonium compounds may also be advantageously combined withiron-sequestering glycoproteins in an antimicrobial composition. Methodsof using a synergistic composition for inhibiting medical device biofilmformation are also disclosed.

U.S. Pat. No. 5,718,899 relates to compositions containing a highconcentration of the full repertoire of immunoglobulins, including IgA,IgM and IgG, are used to combat infections from microorganisms andviruses at a wound, surgical, or burn site, or normal tissue at time ofrisk of infection. The compositions can contain elevated antibody titersfor several specific pathogens including S. aureus, CNS, Enterococci, S.epidermidis, P. aeruginosa, E. coli, and Enterobacter spp, etc. Thecompositions are applied directly to a wound or burn site as anointment, creme, fluid, spray, or the like, prior to vital or bacterialattachment or biofilm formation such that adhesion of the pathogens isinhibited and the pathogens closest to the wound or burn site will bepre-opsonized for phagocytic killing prior to toxin release. Theimmunoglobulins in the composition can be immobilize on a biocompatiblematerial such as collagen, fibrin, hyaluronan, biodegradable polymers,and fragments thereof, which will be placed in-situ at the wound,surgical or burn site. In addition, the immunoglobulins in thecomposition may be coated on the body contacting surface of animplantable device such as a catheter, contact lens, or total joint.These inventive compositions have particular application in preventinginfections.

US 2004/0109852 A1 relates to methods for preventing or removing biofilmon a surface, comprising contacting the surface with an effective amountof a composition comprising one or more acylases and a carrier todegrade a lactose produced by one or more microorganisms, wherein thedegradation of the lactose prevents or removes the biofilm.

U.S. Pat. No. 6,830,745 B1 describes a two component compositioncomprises an anchor enzyme complex to degrade biofilm structures and asecond anchor enzyme component having the capability to act directlyupon the bacteria for a bactericidal effect.

US 2002/0022005 A1 relates to a composition for degrading biofilmstructure associated with cystic fibrosis ²and the debris associatedtherewith comprises an enzyme selected for its ability to dismantle thebiofilm structure, and an anchor molecule coupled to an enzyme to forman enzyme-anchor complex. The anchor molecule is selected for itsability to attach to a surface on or proximal the biofilm structure. Theattachment to the surface permits prolonged retention time of theenzyme-anchor complex where the biofilm structure and associated debrisare present.

WO 02/03998 describes formulation containing between 50 and 750 mg ofmacrolide for delivery by aerosolization to treat infection where thebacteria is suceptible to said macrolide. This patent application alsodescribes methods for treatment of pulmonary infections by a formulation(liquid solution, suspension or dry powder) delivered as an aerosolhaving mass median aerodynamic diameter predominantly inferior to 1 to 5μm.

WO2004/075874 relates to a method of treatment and prevention of acuteor chronic Pseudomonas aeruginosa airway infections through delivery tothe lung endobronchial space, including alveoli, through delivery of aninhalable formulation and consisting in the inhalation of a macrolideantibiotic alone or in combination with another antibiotic.

The problem of resistance of bacterial biofilm to classicalantiinfective agents in respiratory infectious diseases remainscomplete. There is still a need to dispose of a safe and efficientsystem to destroy, disorganize or prevent the formation of such biofilmsin such diseases and/or to restore the activity ofantibiotic/antiinfective drugs. It is also not only desirable to degradebiofilms within a biologic system but it is also necessary to kill thebacterial cells that are released as the biofilm is undergoingdegradation.

OBJECT OF THE INVENTION

The present invention is defined in appended independent claim 1.Preferred embodiments are defined in the dependent claims.

-   -   It is an object of the invention to provide a composition for        inhalation to the lungs which contains a combination of at least        an antimicrobial agent consisting in an aminoglycoside        derivative and a biofilm modifyer which is a Macrolide. The        composition of the invention can be in the form of a dry powder        or in a liquid form, such as a suspension or a solution, or        combination thereof.    -   It is another object of the present invention to provide a        composition for inhalation, active against biofilm, consisting        in the combination of an aminoglycoside and a macrolide, wherein        the concentration (weight/weight) of each active ingredient is        high i.e. superior to 10% of said dry powder composition,        preferably superior to 15%, more preferably superior to 20% of        said composition.    -   It is an object of the present invention to provide a        composition for inhalation wherein the ratio (weight/weight) of        the aminoglycoside and the macrolide is comprised between 0.2        and 5, preferably between 0.5 and 3, more preferably 0.8 and 2.    -   It is another object of the invention to provide a composition        for inhalation wherein the total amount of active drugs        (antibiotic+biofilm modifyer) is comprised between 1 and 50 mg.    -   It is another object of the present invention to provide a        composition for direct administration to the lungs containing an        antimicrobial agent and a biofilm modifyer where the        antimicrobial is an aminoglycoside derivative chosen from        Tobramycin, Kanamycin, Streptomycin, Gentamicin, Amikacin,        Apramycin, Arbekacin, Bekanamycin, Astromycin,        Dihydrostreptomycin, Framycetin, Neomycin, Netilmicin,        Isepamicin, Kanamycin, Micronomicin, Sisomicin or their salts        and derivatives.    -   It is another object of the present invention to provide a        biofilm modifyer selected from the group of macrolides such as        erythromycin, Clarithromycin, Azithromycin, Roxithromycin,        Erythromycin, Telithromycin, Dirithromycin, Flurithromycin,        Josamycin, Kitasamycin, Midecamycin, Dalfopristin, Oleandomycin,        Midecamycin, Pristinamycin, Rokitamycin, Spiramycin, Tilmicosin,        Troleandomycin, Tylosin, Virginiamycin, or their salts and        derivatives    -   It is another object of the present invention to administer the        composition of the present invention as an aerosol or a dry        powder, using a generator system which is a single dose or a        mulitdoses inhaler; dry powder inhalers are often referred to as        DPI.    -   It is another object of the present invention to provide a        composition for inhalation containing an antiinfective agent and        a biofilm modifyer, further containing acceptable pharmaceutical        excipients selected from the group consisting of carbohydrates        or derivatives, lipids derivatives, or other carriers but also        containing sequestring (chelating agents), antioxidants,        stabilizers, buffering agents, surfactants.    -   It is another object of the present invention to provide a        composition for inhalation containing an antiinfective agent and        a biofilm modifyer wherein the carrier is a carbohydrate        selected from the group of sucrose, lactose, monohydrate,        lactose anhydrous dextrose or a combination thereof.    -   It is another object of the present invention to provide a        composition for inhalation containing an antiinfective agent and        a biofilm modifyer wherein the excipients used are lipid        derivatives such as cholesterol, phospholipid derivatives, fatty        acid derivatives or mixtures thereof    -   It is another object of the present invention to provide a        composition for inhalation containing an antiinfective agent and        a biofilm modifyer and a chelating agents such as edetic acid,        citric acid, malic acid, or a salt thereof    -   It is another object of the present invention to provide a        composition for inhalation containing an antiinfective agent and        a biofilm modifyer and a antioxidant agent such as derivatives        of cysteine, ascorbic acid or derivatives, tocopherol        derivatives, propylgallate, parabens derivatives, etc. . . . .    -   It is also an object of the invention to provide a composition        for inhalation efficient against bacterial biofilm consisting in        a combination of an antiinfectious agent which is a        aminoglycoside and a biofilm modifyer which is a macrolide,        further comprising other therapeutically active agents such as        mucolytic, antiinflammatory, and bronchodilator.    -   It is another object of the present invention to provide a        composition for direct administration to the lungs comprising a        combination of antimicrobial which is an aminoglycoside        derivative and a biofilm modifyer which is a macrolide, allowing        to decrease the side-effects and/or the drug-drug interaction,        and/or allowing to increase the compliance and/or the efficacy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the effect of tobramycin (4 μg/ml), clarithromycin(100, 200 and 500 μg/ml) and combinations of tobramycin/clarithromycin(4/100 μg/ml, 4/200 μg/ml and 4/500 μg/ml) on a 12 day biofilm ofPseudomonas aeruginosa.

DETAILED DESCRIPTION OF THE INVENTION

In the present patent, “biofilm modifyer” is defined as a substance ableto destroy, destructure, and disorganize the biofilm and/or to preventor slow down its formation.

For the purpose of the present invention, the terms “antimicrobial”,“antiinfective”, “antibacterial”, and “antibiotic” are synonyms andrefer to substances having a bacteriostatic and for a bactericidaleffect against a given pathogen micro-organism (bacteria, fungi, virus).

The present invention discloses the increase of efficacy ofantimicrobial agents in respiratory infections associated with biofilmby the synergetic combination of at least two active agents consistingof a biofilm modifyer and an antibiotic, administered by inhalation.

Basically, one antibiotic, the aminoglycoside, at least, should beactive against the bacteria contained in the biofilm, while themacrolide shall act on the biofilm for instance by disorganizing it,destructuring it, inhibiting the production of alginate, etc. . . . .The present invention is useful to prevent the formation of biofilm inpatients but also to treat patients with a formed biofilm.

The present invention more precisely consists of a composition—dry orliquid—for inhalation comprising at least one antibiotic from theaminoglycoside group and one antibiotic from the macrolide group, theantibiotic from the macrolide family being active against biofilms(=biofilm modifyer).

The antibiotics from the aminoglycoside group comprise, but are notrestricted to: Tobramycin, Kanamycin, Streptomycin, Gentamicin,Amikacin, Apramycin, Arbekacin, Bekanamycin, Astromycin,Dihydrostreptomycin, Framycetin, Neomycin, Netilmicin, Isepamicin,Micronomicin, Sisomicin or their salts and derivatives. (see Martindale,33^(rd) edition, page 111).

The macrolides from the macrolides group comprise but are not restrictedto: Clarithromycin, Azithromycin, Roxithromycin, Erythromycin,Telithromycin, Dirithromycin, Flurithromycin, Josamycin, Kitasamycin,Midecamycin, Dalfopristin, Oleandomycin, Midecamycin, Pristinamycin,Rokitamycin, Spiramycin, Tilmicosin, Troleandomycin, Tylosin,Virginiamycin, or their salts and derivatives. (see Martindale, 33^(rd)edition, page 112).

There are significant advantages to administer the present compositionof an aminoglycoside and a macrolide directly to the lungs instead oftheir usual route of administration i.e. most often intravenous foraminoglycoside and oral for macrolide. First, the very significantlydecrease of the systemic exposure leads to the decrease of potentiallyvery severe adverse effects of aminoglycoside (nephrotoxicity andototoxicity) and the mild adverse effects of macrolides. Second, theinhalation avoids drug interactions that may occur for some macrolidesthat are metabolized through the cytochrome P450 3A4 (likeclarithromycin). Those interactions may again result in importantadverse effects. Third, the interaction with food is also avoided whenthe composition is inhaled rather than swallowed. And last but notleast, the inhaled route produces very high local concentrations of thedrugs where needed.

The amount of each antibiotic and their respective ratio may vary,depending to the nature of the bacterium to eradicate, the kind ofbiofilm and the kind of infection to treat. The amount of aminoglycosidewill be, in every case, such as to provide, locally, concentrations inaminoglycoside superior to its MIC (Minimal Inhibitory Concentration)against the planktonic bacterium considered. However, the preferredratio (w/w) aminoglycoside/macrolide in the present invention is 0.2 to5, preferably 0.5 to 3, more preferably 0.8 to 2.

The amount of macrolide agent inhaled shall be high enough to affect, insome way, the biofilm. It has to be noted that as the effect ofmacrolide derivatives on the biofilm is mediated through anon-antibacterial mechanism. Therefore, the amounts required to destroythe biofilm by inhalation may be significantly lower than the one neededfor antiinfective activity pre os. Also importantly, the macrolidederivative does not need to possess an antiinfective activity againstthe targeted microorgansim to acts on the biofilm. Nevertheless, it is aanother object of the present invention to provide a compositioncontaining high concentrations (or amounts) of each of theaminoglycoside derivative and of the macrolide derivative i.e. at leastmore than 10%, preferably more than 15%, and more preferably more than20% of the dry powder composition. It is indeed particularly interestingto achieve high lung doses of those therapeutic agents with the minimumamounts of inhalations because it makes the administration easier andmore importantly increase the patient's compliance. It also decreasesthe nominal dose of each active ingredient and thus the adverse effectslinked to these actives. In the present invention, the dry powderinhaler also provides a high Fine Particle Dose (FPD) and Fine ParticleFunction (FPF) when tested in vitro on a Multistage Liquid Impinger (MU,Eur. Pharm, 5^(th) Edition, chapter 2.9.18). The FPD and FPF are theparameters that predict in vivo lung deposition. Briefly, the FPF (%) isdefined as the fraction (expressed of in percent) of the nominal dosepresenting a diameter inferior to 5 μm (maximum diameter of particles tobe able to reach the lungs) and the Fine Particle Dose (FPD) is theamount (in mg) per inhaled unit dose composition presenting a diameterinferior to 5 μm.

High lung deposition of each active ingredient from the composition ofthe present invention will achieve high local concentrations of theantibiotic (generally 5 to 20 times above the Minimal InhibitoryConcentration or MIC) in order to kill the pathogens and high localconcentrations of the biofilm modifyer agent in order to destroy ordestructure rapidly the biofilm.

The DPI composition of the present invention provides with a FPF of atleast 15% of each active ingredient in comparison to the nominal dose,preferably superior to 20%, more preferably superior to 35%.

The preferred ratio (w/w) between the active ingredients(aminoglycoside+macrolides) and the inactive ingredients in dry powdercomposition of the invention, is comprised from 0.2 to 90, preferablyfrom 0.3 to 5, more preferably 0.4 to 2. Alternatively, the compositionsmay be free of excipient (100% of active drugs).

In a preferred embodiment of the present invention, both antimicrobialare present under the form of a dry powder for inhalation agents and areadministered in a fixed combination through inhalation. Said dry powdercompositions may be formulated as a single dose composition i.e. acomposition to be filled individually in capsules or blisters, or as amultidose composition i.e. a composition filled in a device equippedwith a reservoir containing several doses and a metering dose system.

The dry powder composition of the present invention preferably containsthe aminoglycoside derivative in a micronized form and the macrolidederivative in a micronized form. For the purpose of the presentinvention, “micronized” means an average particle size inferior to 20μm, preferably inferior to 10 μm and more preferably inferior to 5 μmwhen measured by laser diffraction for instance. The dry powdercomposition of the present invention may contain more than oneantibiotic and more than one biofilm modifyer

The dry powder composition may further contain other excipients likebuffering agents, surfactants, lubricants, chelating agents orantioxydants, aminoacids. When carbohydrate is used as main inactiveingredient, it has a role of carrier. Then, the preferred process is formanufacturing the composition of the invention is a dry blending of themicronized active ingredients with the non-micronized carrier. In caseof use of a non-micronized carrier, said carrier has preferably a meanparticle size comprised between 50 and 250 μm, preferably between 80 and200 μm, more preferably between 100 and 160 μm. The preferred maincarrier is anhydrous lactose or lactose monohydrate but othermono-disaccharide such as dextrose, xylitol, mannitol, saccharose etc.,may be used. Mixtures of two or more carriers may also be used as wellas mixtures a carrier with other kinds of excipients (lubricants,surfactants, antioxidants, etc.).

The dry powder composition of the invention may contain, in addition tothe main non micronized carrier described hereinabove, a second carrierwhich can be non-micronized or micronized. When this second carrier ismicronized, the preferred mean particle size measured by laserdiffraction is inferior to 20 μm, preferably inferior to 10 μm. Thesecond carrier can be the same chemical entity as the main carrier or adifferent one.

The dry powder composition obtained by dry blending may further compriseexcipients aimed to improve the stability of the composition, theflowability of the powder or the lung deposition of both activeingredients.

Another composition of the invention may contain in addition to themicronized aminoglycoside and the micronized macrolide, a lipidderivative or a mixture of different lipid derivatives as excipients. Inthis case, the preferred process consists in the spray-drying the activeingredients together with the lipid. The spray-drying process requiresthe use of a liquid in which the active ingredients and excipients aresolubilized or in suspension. The solution or suspension is homogeneizedand then spray-dried to obtain a particles in the required mean particlerange i.e. <10 μm, preferably inferior to 5 μm. This spray-dryingprocess is a well known in the pharmaceutical industry and a specificprocess to obtain dry powder composition may, for instance be found inEP 1 674 085 A1.

The preferred lipid excipients are either phospholipids includinganionic phospholipids, cationic phospholipids, zwitterionicphospholipids and neutral phospholipids such as for examplephosphatidylcholine, phosphatidylglycerol, phosphatidyl-inositol,phospatidyl-serine, or non-phospholipids such as glycerol esters (likeglycerol monostearate, glycerol behenate), fatty alcohols (preferablywith C16 or more), fatty acids (preferably with C16 or more), ethers offatty aclohols, esters of fatty acids, hydrogenated oils,polyoxyethylenated derivatives and sterols like cholesterol and itsderivatives. Mixtures of two or more lipid derivatives may also be used.Preferably, a combination of a phospholipid with cholesterol or acholesterol derivative may be used in compositions of the presentinvention.

The lipid excipients may also be combined to other lipidic or nonlipidic excipients like carbohydrate, surfactant, lubricant,antioxidant, chelating agent.

The dry powder composition of the present invention may additionallycontain one or more chelating agent. The chelating agent useful for thepresent invention may include edetic acid (EDTA) or a salt thereof, butother chelating agent such as citric acid, malic acid or their salts maybe used. The chelating agent will preferably be present at aconcentration (w/w) ranging from 0.01% to 5% of the final dry powdercomposition. Combinations of more than one chelating agents may also beused.

The dry powder composition of the present invention may additionallycontain one or more antioxidant agent. Examples of antioxidants that canbe used include derivatives of cysteine like acetylcystein and itssalts, glutathion, carbocystein derivatives or ascorbic acid,derivatives of tocopherol, propylgallate, BHA, BHT.

It is to be noted that the presence of either a chelating agent or anantioxidant agent, or both, may further increase the beneficial effecton the biofilm and may consequently result in a better efficiency thatthe contribution of aminoglycoside and macrolide without these agents.

In a second preferred embodiment, the composition can be in the form ofa liquid, comprising a carrier and both antibiotics (macrolide andaminoglycoside) in suspension and/or solution therein. Nebulizersolutions can be formulated in a similar way to injectable Macrolidesolutions well-known in the art. The liquid carrier is advantageouslywater, or any pharmaceutically acceptable solvent, such as ethanol,dimethylsulfoxide, glycerol, propylene glycol, and mixtures thereof. Theantibiotics in the liquid compositions of the present invention shall bepresent in the same amount ranges as defined supra for the dry powdercompositions.

Example 1 In Vitro Demonstration of the Activity of MicronizedTobramycin+Micronized Clarithromycin on Pseudomonas aeruginosa Biofilm

Biofilms of Pseudomonas aeruginosa—strain PY O₁ were formed according tothe methods described by Ceri et al, the calgary biofilm device: newtechnology for rapid determination of antibiotic susceptibilities ofbacterial biofilms, Journal of clinical microbiology, pp. 1771-1776,1999 and Abdi-Ali et al, bactericidal activity of various antibioticsagainst biofilm-producing Pseudomonas aeruginosa, International Journalof Antimicrobial Agents 27, 196-200, 2006.

PY O₁: is a cystic Fibrosis clinical mucoid strain of Pseudomonasaeruginosa received from the Erasme Hospital, Brussels.

The determination of the minimal inhibitory concentration (MIC) isperformed according to the standard of NCCLS (NCCLS, Methods fordilution Antimicrobial Susceptibility Tests for bacteria that growaerobically; approved standards, sixth edition, M7-A6, vol. 23 no. 2,January 2003.

In the present experiment, the MIC of tobramycin, clarithromycin and thecombination of both antibiotics was first determined on planktonicbacteria (=probacteria i.e. free bacteria not included in a biofilm) toprove that there is no direct additive effect of the active ingredient.The MIC of tobramycin for Pseudomonas aeruginosa is 3.9 μg/ml. The MICof clarithromycin for Pseudomonas aeruginosa could not be determinedsince the results showed that the bacterium is not sensitive to thisantibiotic. The MIC of the combination of tobramycin and clarithromycinis found to be around 3.9 μg/ml. These results, similar to the MIC valuefound for tobramycin alone demonstrate that there is no additionalantibiotic effect of clarithromycin on planktonic Pseudomonasaeruginosa.

In a first attempt to measure the antibiotic activity (MIC) oftobramycin on Pseudomonas aeruginosa when incorporated in a biofilm, aculture of planktonic Pseudomonas aeruginosa was prepared to produce abiofilm during a period of 24 hours. Upon completion of the 24 hours theMIC of tobramycin was measured using these cultures. Surprisingly, itwas found that the Minimum Inhibitory Concentration (MIC) of tobramycinon a 24 hours old biofilm of Pseudomonas aeruginosa was similar to theactivity of tobramycin of planktonic bacteria. In other words,Tobramycin is still active on such a biofilm and there is no need to adda biofilm destroying/destructuring agent.

In a second experiment using the same modus operandi as above, wemeasured the MIC of Tobramycin on a 12 day old Pseudomonas aeruginosabiofilm. This situation is much closer to the situation observed in vivoin chronic respiratory diseases like Cystic Fibrosis. In this case,tobramycin was no longer active against said Pseudomonas aeruginosa.

There exists thus a significant difference between a 1 day old versus a12 days old biofilm: it appears that a biofilm is a living entity thatevolutes from the native to the mature stage. Also these experimentsshall warn researchers that antibiotic activity results obtained fromspecies that form a biofilm may not be taken in consideration unless thebiofilm has had sufficient time to form properly and results found inthe literature have to be taken with precaution.

Effects of Tobramycin, Clarithromycin and Combinations Thereof on 12-DayBiofilm of Pseudomonas aeruginosa.

After having shown that the number of Pseudomonas aeruginosa within thebiofilms was stable after 12 days, the products listed in Table 1 wereadded to the media for the duration of 24 hours. Thereafter the biofilmwas rinsed three times with a 0.01M phosphate buffer adjusted at pH 7.5in order to remove all cells not bound to the biofilm. The microplatewas then placed on ultrasonic bath at 35° C. for 5 minutes to allow thebacteria present in the biofilm to separate from such biofilm. Abacterial count was then performed (number of coloning forming unitCFU/ml). Each experience was done twice and the CFU counting was alsorepeated twice/experience.

The results are shown in Table 1 and FIG. 1. It is concluded thatneither tobramycin 4 μg/ml nor clarithromycin at 100, 200 and 500 μg/mlalone are able to decrease the number of CFU/ml of the 12-day biofilmversus the positive control.

TABLE 1 Effect of tobramycin (4 μg/ml), clarithromycin (100, 200 and 500μg/ml) and combinations of tobramycin/clarithromycin (4/100 μg/ml, 4/200μg/ml and 4/500 μg/ml) on a 12 day biofilm of Pseudomonas aeruginosa. TC C C T/C T/C T/C 4 100 200 500 4/100 4/200 4/500 PC (μg/ml) (μg/ml)(μg/ml) (μg/ml) (μg/ml) (μg/ml) (μg/ml) MIC (CFU × 10⁷) 16 25 14 10 7 31 5 (Low value of MIC is desired.) PC: Positive control (Mueller-Hintonmedium also called CAMHB) T: Tobramycin C: Clarithromycin T/C:Combination Tobramycin/Clarithromycin

To the contrary all the combinations of Tobramycin/Clarithromycin wereable to decrease the number of CFU/ml originated from the biofilm ofPseudomonas aeruginosa with a maximal effect being observed for thecombination TOBRAMYCIN 4 μg/ml+CLARITHROMYCIN 200 μg/ml which shows anumber of CFU/ml of about 10⁷ while tobramycin alone at 4 μg/ml shows anumber of CFU/ml of around 2.5×10⁸. This means a more than 25 timesdecrease in the number of CFU/ml for the combination versus thereference product tobramycin.

It can be seen that enhanced results are determined when at least 100μg/ml clarithromycin is combined with tobramycin, preferably at least200 μg/ml. The efficacy of the mixture decreases somehow forconcentration in clarithromycin grater than 500 μg/ml.

Example 2

A dry powder composition for inhalation of tobramycin and clarithromycinwas formulated using micronized tobramycin supplied by Teva Plantex(Israël). Clarithromycin was supplied by Teva Plantex (Israël) in anon-micronized form. Clarithromycin was then micronized using themicronizer MC-one® (JetpHarma, Switerland). To obtain a product withparticle size suitable to reach the respiratory tract (i.e. 80% ofparticles inferior to 10 μm, and 90% of particles inferior to 5 μm whenmeasured by laser diffraction). The micronisation parameters were apressure of 10 bars in the Venturi, a pressure of 8 bars in the ring anda feeding rate of 5 g/minute. The mean particle size of the micronizedclarithromycin obtained (measured by laser diffraction) was 1.6 μm.

Manufacturing of DPI Composition:

400 g of anhydrous lactose (100-160 μm) were put in a planetary mixertogether with 50 g of micronized lactose monohydrate. The two lactoseswere blended at 40 rpm for 10 minutes. 200 g of micronized tobramycinand 100 g of micronized clarithromycin were added to the mix of lactosesusing the “sandwich technique”, i.e. by alternating the layer oflactoses and the layer of active ingredients to obtain a final mix ashomogeneous as possible. The mix was blended for 10 minutes at a speedof 40 rpm.

Samples were taken from this powder blend to assure both activeingredients were homogeneously blended. 50 mg of the powder mix was thenfilled into number 3 hydroxypropylmethylcellulose (HPMC) capsules. Thesecapsules are ready for use with a dry powder inhaled device such as theMIAT monodose inhaler, or any other suitable capsule based inhalationdevice.

Example 3

Edetic acid in an amount of 0.5% (weight/weight) was added to the blendof example 2. The powder was thereafter filled in a Miat multidoseinhaler device.

Example 4

400 g of anhydrous lactose (100-160 μm) was introduced in a planetarymixer and 150 g of micronized tobramycin, 150 g of micronizedclarithromycin and 20 g of N-acetylcysteinate lysine (as antioxidant)were added using the “sandwich technique”, i.e. by alternating the layerof lactose and the layer of active ingredients to obtain a final blendas homogeneous as possible. The blend was mixed for 10 minutes at aspeed of 40 rpm. The blend was then filled in size 3 hard gelatinecapsules (40 mg of powder/capsule).

Example 5

10 g of micronized tobramycin, 5 g of clarithromycin were dissolved in a80/20 (w/w) water/ethanol mixture. 300 mg of Phospholipon 90H® and 1.2 gof cholesterol were added and dissolved in said solution containing theactive ingredients. The solution was thereafter spray-dried to obtain apowder consisting of micrometric spherical lipidic particles with a veryhigh content in active ingredients. This powder was filled in HPMCcapsules for inhalation (20 mg of powder/capsule).

Example 6

400 g of anhydrous lactose (100-160 μm) was mixed in a planetary mixer(40 rpm for 10 minutes) with 200 g of micronized tobramycin and 200 g ofmicronized clarithromycin. 40 mg of the blend obtained was filled intosize 3 hydroxypropylmethylcellulose capsules. This produced capsuleseach containing 10 mg of micronized tobramycin and 10 mg of micronizedclarithromycin that may be used for inhalation

In Vitro Lung Deposition

The determination of the Fine Particle Fraction (FPF) i.e. the fraction(expressed in percent) of the nominal dose presenting a diameterinferior to 5 μm (maximum diameter to reach the lungs) and the FineParticle Dose (FPD) i.e. the amount (in mg) per capsule presenting adiameter inferior to 5 μm, has been performed on the capsules using theAxahaler device as powder inhaler device. The in vitro lung depositiontest was performed using equipment and conditions as described in theEuropean Pharmacopoeia (5^(th) edition, chapter 2.9.18—apparatus C).This equipment consists of a Multistage Liquid Impinger (MLI) and wasoperated with an air flow of 100 L/min during a period of time of 2.4seconds to simulate inhalation capabilities of patients. Thequantification of the deposition of each drug on each stage of the MLIwas performed by HPLC equipped with a Corona detector. The results arepresented in Table 2.

TABLE 2 FPF (%) and FPD (mg) obtained with the compositions of example 6(MLI 100 L/min) containing 10 mg of tobramycin and 10 mg ofclarithromycin/capsule (n = 3) MLI1 (mg) MLI2 (mg) MLI3 (mg) Mean (mg)SD Tobramycin (mg) Device 1.035 0.938 1.272 1.082 0.17 Throat 0.9290.859 0.771 0.853 0.08 Stage 1 1.760 1.837 1.546 1.714 0.15 Stage 20.614 0.726 0.606 0.648 0.07 Stage 3 1.512 1.818 1.895 1.742 0.20 Stage4 1.598 1.965 2.202 1.922 0.30 Filter 0.664 0.838 0.778 0.760 0.09 FPD(mg) 3.60 4.45 4.72 4.26 0.59 FPF (mg) 35.96 44.50 47.20 42.55 0.06Clarithromycin (mg) Device 1.155 1.048 1.433 1.212 0.20 Throat 1.0251.134 1.223 1.127 0.10 Stage 1 1.338 1.566 1.352 1.419 0.13 Stage 20.664 0.845 0.680 0.729 0.10 Stage 3 1.715 1.891 2.078 1.895 0.18 Stage4 1.162 1.482 1.533 1.392 0.20 Filter 0.478 0.598 0.574 0.550 0.06 FPD(mg) 3.14 3.76 3.98 3.63 0.44 FPF (mg) 31.37 37.56 39.85 36.26 0.04

The FPF of tobramycin and clarithromycin obtained are 42.5% and 36.3%respectively. The FPD/capsule of tobramycin and clarithromycin are 4.26mg and 3.63 mg respectively. Those results clearly demonstrate that thecompositions of the invention allow to reach very high lung depositionof both the antibiotic and the biofilm modifyer. Such high lungdeposition is suitable for use in vivo. Indeed, the volume of epithelialliquid in the lung is generally estimated at about 100 ml. and lungdeposition results show that each capsule of the composition of example6 allows thus to obtain a lung concentration of respectively 42.6 μg/mlof tobramycin and 36.3 μg/ml of clarithromycin.

Example 6

Different compositions (F1 to F5) were manufactured using the blendingprocess as described in example 6.

mg/dry powder composition Active Ingredient F1 F2 F3 F4 F5 Tobramycinbase 20 /  5 15  5 Amikacine / 15  5 / / Clarithromycin 10 10 15 /  5Azithromycin / / / 10 / Anhydrous lactose 20 25 20 20 10 Totalweight/composition 50 50 45 45 20

1-23. (canceled)
 24. A composition for inhalation, comprising at least:(a) an effective amount of an antimicrobial aminoglycoside compound or asalt thereof, and (b) an effective amount of a biofilm modifier which isa macrolide derivative or salt thereof.
 25. The composition of claim 24,in a form of a dry powder.
 26. The composition according to claim 24, ina form of a suspension, solution, or a combination thereof.
 27. Thecomposition of claim 24, wherein a ratio (weight/weight) of each of saidaminoglycoside compound and said macrolide is greater or equal to 10% ofsaid composition.
 28. The composition of claim 27, wherein the ratio isbetween 10-99% of the composition.
 29. The composition of claim 28,wherein the ratio is between 15-90% of the composition.
 30. Thecomposition of claim 24, wherein a ratio (weight/weight)aminoglycoside/macrolide is between 0.2 to
 5. 31. The composition ofclaim 30, wherein the ratio is between 0.3 and
 3. 32. The composition ofclaim 31, wherein the ratio is between 0.8 and
 2. 33. The composition ofclaim 24, wherein a sum of active ingredients represents more than 20%(weight/weight) of the composition.
 34. The composition claim 33,wherein the sum of the active, ingredients is more than 30% of thecomposition.
 35. The composition of claim 34, wherein the sum of theactive ingredients in more than 40% of the composition.
 36. Thecomposition of claim 24, containing at least one or morepharmaceutically acceptable excipients.
 37. The composition of claim 36,wherein at least one of said pharmaceutically acceptable excipients is acarbohydrate or a mixture of two or more carbohydrates.
 38. Thecomposition of claim 37, wherein at least one of said pharmaceuticallyacceptable carbohydrate comprises anhydrous lactose, lactosemonohydrate, mannitol, xylitol, dextrose, saccharose, a cyclodextrincompound or a mixture thereof.
 39. The composition of claim 36, whereinat least one of said pharmaceutically acceptable excipient is a lipidicexcipient.
 40. The composition of claim 39, wherein at least one of saidlipidic excipients comprises cholesterol and derivatives, phospholipid,ethers of fatty alcohols, esters of fatty acids, hydrogenated oils,polyoxyethylenated derivatives, esters of glycerol.
 41. The compositionof claim 40, wherein said composition contains a mixture of cholesterolor a cholesterol compound and a phospholipid or a phospholipid.
 42. Thecomposition of claim 24, further containing one or more chelatingagent(s).
 43. The composition of claim 42, wherein the chelating agentis edetic acid, citric acid, malic acid or a salt thereof.
 44. Thecomposition of claim 24, further containing one or more antioxidant(s).45. The composition of claim 44, wherein at least one antioxidantcomprises a cystein compound, of ascorbic acid compounds, tocopherolcompounds, propylgallate, butylhydroxyanisole, or butylhydroxytoluene.46. The composition of claim 24, wherein the aminoglyscoside compound isTobramycin, Kanamycin, Streptomycin, Gentamicin, Amikacin, Apramycin,Arbekacin, Bekanamycin, Astromycin, Dihydrostreptomycin, Framycetin,Neomycin, Netilmicin, Isepamicin, Micronomicin, Sisomicin or apharmaceutically acceptable salt thereof.
 47. The composition of claim24, wherein the macrolide compound is a biofilm modifier macrolide or anantibiotic macrolide, and comprises of Clarithromycin, Azithromycin,Roxithromycin, Erythromycin, Telithromycin, Dirithromycin,Flurithromycin, Josamycin, Kitasamycin, Midecamycin, Dalfopristin,Oleandomycin, Midecamycin, Pristinamycin, Rokitamycin, Spiramycin,Tilmicosin, Troleandomycin, Tylosin, Virginiamycin, and thepharmaceutically acceptable salts.
 48. The composition of claim 24,wherein the aminoglyscoside compound is Tobramycin or a salt thereof andthe macrolide compound is clarithromycin or a salt thereof, saidcomposition being free of another antimicrobial agent.
 49. Thecomposition of claim 24, wherein said composition is free of excipients.50. The composition of claim 25, wherein said dry powder composition isfilled in pharmaceutically acceptable capsules.
 51. The composition ofclaim 48, wherein said pharmaceutically acceptable capsule contains, asmain polymer, gelatin, hydroxypropylcellulose or starch.
 52. Thecomposition of claim 25, wherein the said dry powder composition isfilled into a multidose dry powder inhaler device.